In general, the purpose of this research is to identify the various lysosomal peptide hydrolases in the connective tissue of bovine dental pulp, and to define their role in promoting the degradation of connective tissue macromolecules. More specifically, one such enzyme, tentatively identified as dipeptidyl aminopeptidase II, was recently found to be abundant in dental pulp, to have a lysosomal distribution in the fibroblasts of that tissue, and to have a range of hydrolytic activity that included the scission of prolyl bonds, as well as alanyl bonds, at acid pH. A major effort will be made to purify and characterize this enzyme and to define its substrate specificity on a range of natural and synthetic peptides, especially ones having proline-rich sequences (as in collagen) and alanine-rich sequences (as in elastin). The properties of the enzyme purified from bovine dental pulp will be compared with an authentic sample of DAP II purified from dental pulp. Through the use of a histochemical substrate (now available), or a peroxidase-labeled antibody, ultrastructural studies will be performed to locate dipeptidyl aminopeptidase II in dental pulp and in other oral tissues as well. An attempt will be made to apply this technique together with quantitative fluorometric assay methods, to the enzyme analysis and ultrastructural examination of inflamed periodontal tissues of human origin. Similar biochemical and cytochemical studies will be conducted with other lysosomal enzymes, for example, cathepsin B1. This potent, thiol-activated proteinase was recently demonstrated in this laboratory to be present in dental pulp. Since cathepsin B1 purified from bovine spleen has been reported to attack native collagen, an effort will therefore be made to characterize the substrate specificity of the purified dental pulp enzyme, particularly in regard to its collagenolytic and proteolytic activities.